Accounting Problems Under the Excess Profits Tax

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV- 1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8(+) T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.Funding Agencies|Research Council of Norway; Odd Fellow</p

Increased Generation of HIV-1 gp120-Reactive CD8(+) T Cells by a DNA Vaccine Construct Encoding the Chemokine CCL3

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV- 1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8(+) T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.Funding Agencies|Research Council of Norway; Odd Fellow</p

Characterization of the gp120-containing vaccibodies.

<p>(A, B) SDS-PAGE (4–20% Tris-Glycine gel) and Western blotting of supernatants harvested from HEK293 cells transfected with DNA encoding the various vaccibodies indicated by their targeting unit. (Mock = supernatant from HEK293 transfected with DNA encoding a fluorescent protein). The supernatants were either left untreated (−ME) (A) or reduced with mercaptoethanol (+ME) (B) prior to SDS-PAGE and blotting. The vaccine proteins were detected by anti-human IgG antibodies (clone HP6017) and chemiluminescence. (C) The gp120-containing vaccibodies were produced and secreted by transfected cells. ELISA of supernatants harvested from HEK293 cells transfected with DNA encoding the vaccine constructs indicated in the figure. The antibodies used as coat and detection in the ELISAs are indicated above each graph. Similar results were obtained in multiple experiments, and one representative experiment is presented. (D) CCL3 inserted into the vaccibody format is chemotactic. Supernatants containing the indicated vaccine proteins (left graph) or recombinant CCL3 (right graph) were added to the bottom wells of Transwell plates. 600 000 Esb-MP cells (a murine T cell line that expresses CCR1 and CCR5) were added to the top wells and the number migrating through the membrane was determined by flow cytometry after 2 h. Representative results from one out of two experiments are shown. (E) αMHCII but not αNIP inserted into the gp120-containing vaccibody binds to MHC class II. Supernatants harvested from transfected HEK293 cells were used to stain MHC class II Eβ^dEα^k-transfected mouse fibroblasts. The bound vaccine proteins were detected by a C_H3-specific antibody (clone HP6017) and fluorescence microscopy. Scale bar, 10 µm.</p

Vaccibody induced long-lasting gp120-reactive serum antibodies which can neutralize HIV-1.

<p>BALB/c mice were immunized once with DNA encoding the indicated vaccine constructs. DNA (25 µg DNA diluted in 50 µl NaCl) was injected into the quadriceps before electroporation. (A) Levels of gp120-reactive IgG-antibodies in mice sera harvested 56 days after immunization with the indicated vaccibodies. The levels are indicated by endpoint titers and were determined by ELISA using gp120 as coat. n = 5 mice/group. (B) Effects of targeting on the level of gp120-reactive IgG-antibodies in sera. Sera were harvested at the indicated time points, and the amounts of gp120-reactive antibodies were determined by ELISA using gp120 as coat. Anti-gp120 IgG levels differed significantly between the two vaccine groups at day 28, *p = 0.003, n = 3 to 4 mice per group. The experiment was performed twice and showed similar results. (C) Titers of gp120-reactive IgG-serum antibodies 10 months after immunization with DNA (2 µg diluted in 50 µl NaCl) encoding vaccibodies or gp120. Sera were analyzed by ELISA using gp120 as coat. n = 4 mice per group. (D) αMHCII-gp120 induced antibodies that neutralized HIV-1. Sera harvested 10 or 15 months after injection of DNA or NaCl were incubated together with Jurkat T cells and HIV-1. A murine IgG1 antibody (clone P4/D10) that can neutralize HIV-1IIIB through recognition of amino acid 304–323 of gp120 was used as positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104814#pone.0104814-Broliden1" target="_blank">[49]</a>, and its inhibition of HIV-1 infection was set to 100%. Percentage inhibition by the mice sera is relative to the positive control. n = 4 mice per group. In A–D: mean values and SEM are presented.</p

Vaccine with CCL3 inserted in the targeting unit induced higher number of gp120-reactive T cells <i>in vivo</i>.

<p>BALB/c mice were immunized by one single DNA injection in each quadriceps muscle in the absence of electroporation (25 µg DNA in 50 µl NaCl per quadriceps). (A, B) IFNγ-ELISpot of splenocytes harvested four weeks after immunization with the indicated vaccine constructs. The splenocytes were incubated together with either (A) P18, an MHC class I-binding peptide derived from gp120, or (B) a pool of peptides derived from gp120 (overlapping 15 amino acid peptides). n = 4 mice per group, *0.05>p>0.01; **p = 0.01; ***p<0.01 (analyzed by one-way ANOVA and Šidák method). (C) Kinetics of P18-reactive CD8⁺ T cells in blood. Blood was collected at the indicated time points and P18-reactive CD8⁺ T cells were identified by staining with MHC class I/P18-tetrameric H-2D^d complexes and flow cytometry. Percentage tetramer positive cells were calculated from gated CD8⁺ T cells. n = 4 mice per group. In A–C: mean values and SEM are presented.</p

Schematic drawing of gp120-containing vaccibodies.

<p>(A) The vaccibody format is a homodimer which consists of three functional units: the targeting, the dimerization, and the antigenic unit. The chemokine CCL3 and the Ig variable regions specific for MHC class II (denoted αMHCII) were utilized to target APCs. The variable regions were assembled in a single chain variable fragment-format (scFv). (B) Vaccibodies without the ability to target APCs, indicated as non-targeted vaccibody, were generated by use of a mutant of CCL3 (CCL3C11S) which does not bind receptor and variable regions specific for the hapten NIP (denoted αNIP). These variable regions were also inserted into the scFv-format.</p

Effects of electroporation and targeting molecules on the cellular immune response.

<p>Immunizations of BALB/c mice (25 µg DNA in 50 µl NaCl per quadriceps) were performed without (−EP) or with electroporation (+EP). (A) Electroporation increased the amount of luciferase at the injection site. DNA encoding luciferase was delivered intramuscularly in the quadriceps without (−EP) or with electroporation (+EP), and the expression of luciferase was measured by <i>in vivo</i> imaging and bioluminescence (p/s: photons/second) at different time points after injection. The amount of luciferase differed significantly between the two groups from day 4, *p≤0.02, n = 3–4 muscles/group. (B, C) Cellular immune responses induced by the vaccibodies delivered by intramuscular DNA-injection without (−EP) or with electroporation (+EP). Splenocytes were harvested four weeks after immunization and incubated together with the MHC class I, P18-peptide (B) or a pool of overlapping peptides from gp120 (C) in IFNγ-ELISpot assay. n = 8 mice per group for CCL3-gp120 and n = 4 mice per group for αMHCII-gp120 and αNIP-gp120. In A–C: mean values and SEM are presented. B and C, for the groups of control mice (NaCl delivered with or without EP, and re-stimulated with P18 or gp120 peptides) the mean number of IFNγ-SFC/10⁶ splenocytes varied from 37 to 47 and is substracted from the number of spots in the respective samples from vaccibody-immunized mice.</p

Antibodies utilized in ELISA, Western blotting, and immunostaining.

<p>m, mouse; h, human; AP, alkaline phosphatase.</p

Antibody and T cell responses following vaccination with OVA targeted to MHC class II molecules or CCR1/3/5.

<p>(a,b) Supernatants of 293E cells transfected with the indicated plasmids were tested for secreted proteins in ELISA (a) and examined by Western blotting with anti-OVA mAb under reducing (+ME) or non-reducing (-ME) conditions (b). Vaccine proteins are indicated below lanes. (c-f) Mice were immunized once i.d. with 25 µg DNA/EP, as indicated. (c-e) Sera were assayed for total IgG (c), IgG1 (d) or IgG2a (e) against OVA. (f) Splenocytes collected at day 14 post immunization were stimulated <i>in vitro</i> with OVA protein or controls as indicated, and analyzed by an IFNγ EliSpot. *indicates p<0.008 and **p<0.002.</p

Characterization of fusion vaccine proteins.

<p>a) Schematic overview of homodimeric vaccine proteins. The fusion proteins consists of HA antigen connected to a targeting unit via a shortened Ig hinge and a dimerizing human γ3 CH3 domain and Ig hinge. As targeting units we used a scFv directed against the MHC class II molecule I-E^d (αMHCII-HA), or the mouse chemokine MIP-1α (MIP-1α-HA). For non-targeted controls, a scFv directed against the hapten NIP (αNIP-HA), or a mutated MIP-1α (MIP-1α(C11S)-HA), replaced functional targeting units. b) Supernatants of transfected 293E cells were examined by Western blotting with anti-HA mAb under reducing (-ME) or non-reducing (+ME) conditions. Vaccine proteins are indicated below lanes, and MW by arrows. c) Binding of vaccine proteins to anti-C_H3 mAb in Sandwich ELISA, followed by detection with an anti-HA mAb. d) Binding of vaccine proteins to MHCII I-E^d-transfected L cell fibroblasts. Vaccine proteins were detected by anti-HA mAb. e) Supernatants of 293E cells transfected with MIP-1α-HA or the mutated counterpart (C11S) were examined for chemotaxis. Recombinant human MIP-1α(rLD78β) was included as positive control. Chemotactic index is shown. f, g) Binding of vaccine proteins to CD11b⁺ BALB/c splenocytes.</p